Vascular homeostasis is an essential biological process involving multiple cell types and signaling pathways. Numerous factors implicated in vessel function (i.e. shear stress, cytokines, growth factor stimuli) communicate through PI3K/Akt, a key signaling intermediate. Endothelial cells (EC) express predominantly Akt1, the major isoform involved in regulation of cardiovascular function. Our previous work suggested that atherosclerotic lesion expansion in Akt1-/-;ApoE-/- mice was likely from vessel origin, implying disease progression is dictated by EC or smooth muscle cell (SMC) lineage. However, SMC-specific Akt1 deletion does not affect atherosclerotic lesion formation (unpublished, collaborator), designating the endothelium as a critical mediator of plaque development. AIM 1 will therefore implement EC-targeted Akt1 conditional mouse models to substantiate the importance of endothelial Akt1 expression in cardiovascular disease outcome. We also report impaired eNOS phosphorylation in Akt1-/-;ApoE-/- mice, paralleling the emerging importance of eNOS functionality for cardiovascular homeostasis. While previous genetic studies signify eNOS as an Akt1 substrate, the definitive role of Akt1-directed eNOS activation during atherogenesis remains to be clarified. AIM 2 will extend these genetic studies to directly test the causal role o Akt1-dependent eNOS-S1176 phosphorylation on plaque formation. Use of global Akt1-null mice expressing either the `constitutively-active' eNOS (S1176D; Akt1-/-; ApoE-/-) or `less-active' eNOS (S1176A; Akt1-/-; ApoE-/-) will delineate the role of EC-derived NO production on atherosclerotic lesion development. Lastly, our recent Akt phospho-proteomic analysis of endothelial lysates indicates a number of new Akt substrates with potential influence on vascular function and/or permeability (e.g. VE-PTP, NUP93). AIM 3 will integrate the results from our high-throughput, phospho-proteomic array to provide mechanistic insight on previously unexplored Akt-directed methods of endothelial regulation. Understanding the kinase-substrate relationship between Akt and the newly identified target proteins will undoubtedly broaden our current perspective of Akt1 function and the implications on cardiovascular function. Overall, this proposal aims to decipher the role of endothelial Akt1 expression/activity on both EC physiology and cardiovascular homeostasis using several molecular, cellular, and genetic approaches. Training: This proposal outlines a 5-year career development plan to facilitate the candidate's transition from a mentored postdoctoral fellow to an independent principle investigator. The candidate is currently in her 4th year of post- doctoral training where she continues to participate in regular seminars and present at scientific meetings. This application builds upon the candidate's background in smooth muscle biology by providing specialized focus on endothelial physiology and vascular function. The research proposed herein will be conducted under the continued mentorship of Dr. William C. Sessa, Ph.D. (Yale University, Department of Pharmacology), a recognized leader in the field of endothelial biology and atherogenesis. Several key collaborations have also been established for scientific guidance related to this proposal, including Dr. Carlos Fernandez-Hernando, Ph.D., an expert in cholesterol and lipoprotein metabolism, and Dr. Patrick Lusk, Ph.D., an expert on nucleoporins and nuclear compartmentalization. The results from this study will not only advance our understanding of endothelial PI3K/Akt signaling, but also provide an invaluable mentoring experience to create the foundations of a future independent researcher.